Selfemulsifiable formulation having enhanced bioabsorption and immunosuppression activities

ABSTRACT

A selfemulsifiable formulation comprising a lipophilic system consisting of a medium chain triglyceride of caprylic acid and capric acid, and of labrasol, wherein labrasol also serves as a surfactant, which is combined with other selected surfactants, like cremophore RH 40 and/or polysorbate 80 and wherein the formulation also comprises immunosuppression agent, essentially cyclosporine, hydrophilic agent preferably ethanol, antioxidant preferably alpha-tocopherol and preservative preferably benzyl alcohol. The formulation is prepared by dissolving immunosuppression agent in hydrophilic agent followed by entrapping with lipophilic agent and subsequent treatment with surfactants, preservative and anioxidant. The formulation is filled in a soft-gelatin shell capable of rupturing in less than 10 minutes to deliver the formulation in an upper part of the gastrointestinal tract, wherein it forms thermodynamically stable oil in water microemulsions in situ to have enhanced bioavailability and bioabsorption of immunosuppression agent, which can show its enhanced immunosuppression activites thereby.

TECHNICAL FIELD OF INVENTION

The present invention relates to a formulation, particularly to aselfemulsifiable formulation for oral administration having enhancedbioabsorption and immunosuppression activities, more particularly itrelates to a selfemulsifiable formulation for early delivery of drug,even more particularly it relates to a selfemulsifiable formulation,which will form thermodynamically stable oil in water emulsionpreferably in upper part of gastrointestinal tract and facilitate theearly delivery of drug, particularly of the immunosuppression agent,still more particularly it relates to a selfemulsifiable formulation notonly having the improved bioavailability and bioabsorption but also hasimproved capability to release the drug in reduced time with reducedtoxicity and variability that is inter and intra patient bioabsorptionvariability. The present invention is particularly relates to aselfemulsifiable formulation, which facilitates increased solubility,transport rate, bioavailability and bioabsorption of an agent havingimmunosuppression activity.

BACKGROUND ART OF INVENTION

The immunosuppression activity of a drug acting as an immunosuppressionagent is achieved by inhibiting the growth and differentiation ofT-cells. Such immunosuppression agents also have other pharmacologicalactivities like anti-inflammatory and/or antiparasitic, in particularantiprotozoal, like antimalerial activities. The commonly usedimmunosuppression agents include cyclosporine. There are manycyclosporines known in the art, like cyclosporine A, cyclosporine B,cyclosporine C, cyclosporine D, cyclosporine E etc. The cyclosporine Ais preferably used in the clinical field due to its provenpharmacological activity and clinical indication and effectiveness.

This immunosuppression agent, that is, cyclosporine A has been founduseful in various other areas, like in auto immune diseases,inflammatory conditions, particularly in inflammatory conditions with anaetiology including an autoimmune component like arthritis.

Further, this immunosuppression agent is applicable in rheumatoidarthritis, arthritis chronica, and progredientic and arthritisdeformana. Further, this immunosuppression agent is also applicable inrheumatic diseases.

The immunosuppression therapy using this immunosuppression agent hasbeen proposed or applied in autoimmune hematological disorder, likehemolytic anemia, aplastic anaemia, pure red cell anaemia and idiopathicthrombocytopaenia, systemic lupus erythematosus, dematomyositis, chronicactive hepatitis, myasthenia gravis, psoriasis, Steven-Johnson syndrome,idiopathic spure, autoimmune inflammatory bowel disease includingulcerative colitis and crohn's disease, endocrine opthalmopathy, Gravesdisease, sarcoidosis, multiple sclerosis, primary billiary cirrhosis,juvenile diabetes, like diabetes mellitus Type I, anterior and posterioruveitis, and keratoconjuncativities sicca an vernalkeratoconjuncativities, intestinal lung fibrosis, psoriatic arthritisand glomerulonephritis—with or without nephrotic syndrome, likeidiopathic nephrotic syndrome or minimal change nephropathy.

Therefore, this immunosuppression agent is widely acceptableimmunosuppression agent. This agent is made available in the form ofpharmaceutical formulation. The clinical acceptance of such formulationscomprising of this immunosuppression agent has suffered due to lowsolubility and low transport rate and delayed bioavailability andbioabsorption of the immunosuppression agent.

Therefore, in recent past the research has been directed to improve itssolubility and transport rate. In addition efforts have been on toimprove its early bioavailability, particularly in the upper part of thegastrointestinal tract and bioabsorption.

Various formulations, comprising this immunosuppression agent as one ofthe essential ingredients, have been developed and made available.Although there are many formulations to form microemulsions, but many ofthem do not have satisfactorily acceptable bioavailability andbioabsorption. The formation of microemulsions of the clinicallyacceptable particle size that is of less than 200 mm, particularly ofless than 100 nm, is one of the desired requirements for the formulationto be clinically acceptable. Therefore, the efforts are still on todevelop new formulations, particularly the formulations which will havebetter bioavailability, particularly in the upper part of thegastrointestinal tract and better bioabsorption and at the same timewill have better solubility and reduced variability, that is inter andintra patient bioabsorption variability, and form the microemulsions ofthe clinically acceptable particle size.

The another parameter controlling the applicability of formulations ofthe immunosuppression agent is its manner of administration. Theformulation comprising of the immunosuppression agent, particularly ofthis immunosuppression agent is generally administered after filling itin a soft or hard shell, known as capsule, or in the form of solutionfor oral administration. The solution form of formulation of theimmunosuppression agent is taken after dilution with flavored milk orfruit juice. The mixing with milk or the juice forms the emulsion,particularly microemulsions of varying particle sizes, generally varyingabove 100 nm, preferably varying above 150 nm. The preferred form ofadministration of the formulation of this immunosuppression agent isafter filling the formulation in a shell, which may be soft or hardshell.

The major problem arises, when the immunosuppression agent or itsformulation is administered after filling in a hard or soft-shell. Ithas been generally observed that, the availability of theimmunosuppression agent will depend upon the rupture time of the shell.

The known formulations of the immunosuppression agent, which are madeavailable in the shell, have rupture time of shell varying from 12minutes to 15 minutes or above. The problem arises due to this longerrupture time, which delays the availability of the immunosuppressionagent, which in-turn effects its bioabsorption. The desired rupture timeof the shell in order to make the availability of the immunosuppressionagent at an early time, preferably in the upper part of thegastrointestinal tract is less than 12 minutes, preferably less than 10minutes.

Need of Invention

Therefore, there is a need to have a formulation, particularly aselfemulsifiable formulation for oral administration, which can overcomeall or some of the disadvantages and limitations of the prior art, asdescribed herein above and more particularly of a selfemulsifiableformulation, which facilitates increased solubility, transport rate,bioavailability and bioabsorption of the immunosuppression agent.

OBJECTS OF INVENTION

This is the main object of the present invention to make a completedisclosure of a formulation, particularly of a selfemulsifiableformulation for oral administration, which can overcome all or some ofthe disadvantages and limitations of the prior art, as described hereinabove and more particularly of a selfemulsifiable formulation, which canfacilitate the increased solubility, transport rate, bioavailability andbioabsorption of the immunosuppression agent.

Another object of this invention is to propose for a selfemulsifiableformulation capable of making early bioavailability of theimmunosuppression agent, particularly in the upper part of thegastrointestinal tract.

Still another object of this invention is to propose a selfemulsifiableformulation, which can satisfactorily meet the clinical requirements.

Still further an object of the present invention is to disclose aselfemulsifiable formulation, which can form microemulsions of particlesize less than 200 nm, preferably less than 100 nm.

Yet another object of this invention is to propose a selfemulsifiableformulation, which can administered orally after filling in a soft orhard shell, or in the form of solution.

This is further an object of this invention to disclose aselfemulsifiable formulation, which can form microemulsions of particlesize less than 200 nm, preferably less than 100 nm and a clear solutionwhen administered orally in the form of microemulsions, mixed with fruitjuice, milk or any aqueous medium.

This is still an object of this invention to disclose a selfemulsifiableformulation, which can form microemulsions of particle size less than200 nm, preferably less than 100 nm when administered orally afterfilling in a soft or hard shell.

This is yet an object of this invention to disclose a selfemulsifiableformulation, which on administration in a shell, particularly in a softshell is made available at an early time, preferably in the upper partof the gastrointestinal tract in less than 12 minutes, more preferablyin less than 10 minutes, by rupturing the shell in desired time of lessthan 12 minutes.

This is still an object of this invention to disclose a selfemulsifiableformulation, which has better solubility and reduced variability, thatis inter and intra patient bioabsorption variability.

Still another an object of this invention is to disclose aselfemulsifiable formulation, which can be stored in the tropicalcountries for a longer time and can forms thermodynamically stable oilin water microemulsions in-situ, which are stable for more than 24 hrs.

Yet another an object of this invention is to disclose aselfemulsifiable formulation and the method of preparation thereof.

Further objects, advantages and preferred embodiments of the presentinvention will be more apparent from the following description when readin conjunction with the accompanying drawings, which are not intended tolimit the scope of the present invention.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the phase diagram of the selfemulsifiable formulation inaccordance to the preferred embodiments of the present invention.

FIG. 2 shows the phase diagram representing the relative concentrationof oil phase, water phase and mixture of surfactants of theselfemulsifiable formulation in accordance to the preferred embodimentsof the present invention.

FIG. 3 shows the comparative dissolution profile of experiments XIII,XIV and XV of the selfemulsifiable formulation in accordance to thepreferred embodiments of the present invention and of Sandimmun Neoral,which is taken as standard.

FIG. 4 shows the percent amount of normalized particle size inexperiment XV, the best preferred experiment of the selfemulsifiableformulation in accordance to the preferred embodiments of the presentinvention.

FIG. 5 shows the mean plasma concentration of the immunosuppressionagent after administration of formulation, after filled in soft-shell,of experiment XV, the best preferred experiment of the selfemulsifiableformulation in accordance to the preferred embodiments of the presentinvention.

FIG. 6 shows the compositions of formulations prepared in accordance tothe preferred method of the present invention.

BRIEF DESCRIPTION OF INVENTION

Accordingly this invention provides a complete disclosure of aformulation, particularly of a selfemulsifiable formulation for oraladministration, which has enhanced bioabsorption and immunosuppressionactivities, more particularly of a selfemulsifiable formulation forearly delivery of drug, even more particularly of a selfemulsifiableformulation, which will form thermodynamically stable bil in wateremulsion preferably in upper part of gastrointestinal tract andfacilitate the early delivery of drug, particularly of theimmunosuppression agent, still more particularly of a selfemulsifiableformulation not only having the improved bioavailability andbioabsorption but also the improved capability to release the drug inreduced time with reduced toxicity and variability, that is inter andintra patient bioabsorption variability.

In accordance to the present invention a selfemulsifiable formulation isdisclosed, which facilitates the increased solubility, transport rate,bioavailability and bioabsorption of an agent, particularly of animmunosuppression agent, wherein said formulation essentially comprisesof immunosuppression agent, hydrophilic agent, lipophilic agent, one ormore of surfactants, antioxidant and preservative. In accordance to oneof the preferred embodiments of the present invention the formulation ismade available in a shell; preferably soft shell, wherein the said shellessentially comprises of gelatin, glycerin, water, one or more ofpreservatives and one or more of colorants.

The formulation of the present invention can be prepared by any knownmethod. In accordance to the preferred embodiment of the presentinvention the preferred method, for preparation the presently disclosedselfemulsifiable formulation comprises of dissolution ofimmunosuppression agent in hydrophilic agent followed by entrapping ofsolubilised immunosuppression agent with lipophilic agent, which in-turnis followed by treatment of oil entrapped solubilised form of drug withone or more of surfactants and the resulted solubilised drug entrappedwith oil and one or more of surfactants is treated with preservative andantioxidant.

DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS OF INVENTION

In accordance with this invention a selfemulsifiable formulation fororal administration, as described herein above, is disclosed, whereinsaid formulation essentially comprises of immunosuppression agent,hydrophilic agent, lipophilic agent, one or more of surfactants,antioxidant and preservative, wherein immunosuppression agent ispreferably lactam macrolide having immunosuppression activity;hydrophilic agent is selected from a group consisting ofpharmaceutically acceptable C₁₋₅ alkyl, tetrahydrofuryl diether,tetrahydrofuryl partial ether, low molecular weight monooxy-alkane-diol,low molecular weight polyoxy-alkane-diol, 1,2-propyleneglycol, ethanol;lipophilic agent is selected from a group consisting of medium chainmonoglycerides, medium chain diglycerides, mixed esters of saturatedfatty acids, like caprylic and/or capric acids, medium chaintriglycerides of caprylic and/or capric acids; one or more of surfactantis selected from a group consisting of hydrogenated vegetable oils,polyoxyethylene sorbitan fatty acids, transesterified caprylic and/orcapric glycerides; antioxidant is selected from a group consisting ofalpha-tocopherol, ascorbyl palmitate, butyl hydroxy anisole, butylhydroxy toluene, propyl gallate; preservative is selected from a groupconsisting of ethanol, benzyl alcohol.

In accordance to the most preferred embodiment of the present invention,it has been surprisingly found that to have particular stablemicroemulsions of clinically acceptable particle size with enhancedbioavailability and reduced variability in inter and intra-patient doseresponse, are obtained by using novel lipophilic agent of the presentinvention. The most preferred lipophilic agent of the present invention,which may also be referred to, as drug or immunosuppression agentcarrier is medium chain triglyceride of caprylic acid and capric acid,named to as labrafac lipophile. In accordance to the preferredembodiment of the present invention the medium chain triglyceride ofcaprylic acid and capric acid, which is used as lipophilic agent in thepresently disclosed formulation is obtained by any known method, like byesterification of glycerol by caprylic acid and capric acid at hightemperature. The medium chain triglyceride of caprylic acid and capricacid, which is selected as lipophilic agent has specific gravity ofabout 0.93 to 0.96, refractive index of about 1.44 to 1.452, acid valueless than about 0.2, saponification value of about 310 to 360 and iodinevalue less than about 1 and water content less than about 0.5.

In accordance to one of the preferred embodiments of the presentinvention immunosuppression agent is cyclosporine, particularlycyclosporine A having immunosuppression activity; hydrophilic agent isadditionally selected from a group consisting of pharmaceuticallyacceptable lower (C₁₋₄) alkanols, like ethanol; alkylene glycolmonoalkyl ethers, like diethylene glycol monoethyl ethers, franscutol,glycofural (known as tertrahydrofuryl) alcohol polyethylene glycolether. The preferred hydrophilic agent is lower alkanol, preferablyethanol.

The preferred lipophilic agent is medium chain triglyceride of caprylicacid and capric acid.

The surfactant, one or more, is/are additionally selected from a groupconsisting of saturated polyglycolysed C₈ to C₁₀ glycerides, liketransesterified caprylic and/or capric glycerides, particularly PEG-8caprylic and/or capric acid glyceride exhibiting specific gravity ofabout 0.930 to 0.960, refractive index of about 1.44 to 1.452, acidvalue less than about 0.2, saponification value of about 85 to 105,peroxide value less than about 6.0. free glycerol content less thanabout 5.0%, ethylene oxide content of about 1.0 ppm, water content lessthan about 1.0%, capric acid less than about 2.0%, caprylic acid (C₈)about 50 to 80%, capric acid (C₁₀) about 20 to 50%, capric acid (C₁₂)less than about 3% and myristic acid (C₁₄) less than about 1.0%,hydrophilic liophilic balance, referred to as HLB, value of about 14;polyoxyethylene sorbitan fatty acid esters, like polysorbate 20,polysorbate 40, polysorbate 80, more preferably polysorbate 80;polyoxyethylene castor oil derivatives, like cremophor RH 40, cremophoreEL, preferably cremophore RH 40.

The preservative, to protect the formulation during storage and use fromany microbial growth, particularly in tropical region, is selected froma group consisting of ethanol, benzyl alcohol.

In accordance to the present invention the immunosuppression agent istaken in an amount of about 2 to 10%, preferably in an amount of about 5to 10%. It is taken in ratio of about 1:05 to 1:2-5 more preferably ofabout 1:1.25 by weight with respect to hydrophilic agent. Thehydrophilic agent is taken alone or in combination. The hydrophilicagent is taken in weight ratio of about 1:1 to 1:6. The preferably usedhydrophilic agent is ethanol, which is taken in the ratio of about 1:0.5to about 1:2.5 with respect to immunosuppression agent. The otherpreferred hydrophilic agent is diethylene glycol monoethyl ethers. Thelipophilic agent, preferably medium chain triglyceride of caprylic andcapric acids is present in the system in the ratio of immunosuppressionagent to lipophilic agent of about 1:0.5 preferably of about 1:1.5 or1:1.7 more preferably of about 1:4 by weight for entrapment ofsolubilised immunosuppression agent, particularly cyclosporine, moreparticularly cyclosporine A. The other lipophilic agents used in thepresent invention include capmul MCM and crodamal GTCC, which are usedin the ratio of about 1:0.5 to 1:4 by weight with respect to hydrophilicagent. Still other lipophilic agent of the present invention includescombination of labrafac and labrasol are taken in the ratio of about1:3, preferably of about 1:3.5, more preferably of about 1:4 by weight.In accordance to the present invention labrasol acts as surfactant.

The surfactants, in accordance to the present invention, includetransesterified caprylic and/or capric glyceride, like labrasol, whichis taken in the ratio of about 2:1, preferably of about 3:1, morepreferably of about 6:1 by weight with respect to lipophilic agent ofthe present invention. In accordance to one of the preferred embodimentsof the present invention the labrasol is used in combination with one ormore of other surfactants, like cremophore RH 40 in the ratio of about1:1, preferably of about 2.5:1 by weight or with polysorbate 80 in theratio of about 2:1, preferably of about 4.5:1 by weight. In accordanceto one of the preferred embodiment of this invention the combination oflabrasol, cremophor RH 40, polysorbate 80 is taken in the ratio of about4:1:1.5 by weight respectively which gives clear translucentmicroemulsions with bluish tinge and particle size less than 100 nm.

The preservative, in accordance to the present invention, includesbenzyl alcohol in the amount of about 0.5 to 1% by weight.

The antioxidant, in accordance to the present invention, includesalpha-tocopherol in the amount of about 0.0007 to 0.00009.

The formulation of the present invention can be prepared by any knownmethod. The preferred method, in accordance to one of the preferredembodiments of this invention comprises of following steps;—

-   a) dissolution of immunosuppression agent in hydrophilic agent,-   b) entrapping of solubilised immunosuppression agent with lipophilic    agent,-   c) treatment of oil entrapped solubilised form of drug with one or    more of surfactants,-   d) treatment of solubilised drug entrapped with oil and one or more    of surfactants with preservative and antioxidant

In accordance to the preferred method of the present invention the firststep involves dissolution of selected amount of immunosuppression agentin selected amount of hydrophilic agent. The concentration ofhydrophilic agent is optimized to such a level in the present invention,that it will keep the immunosuppression agent in solubilized form tillthe formulation shelf life. The solubilisation step of the presentlydisclosed method is followed by entrapping with selected amount ofpresently disclosed lipophilic agent, which acts as carrier during theabsorption of the drug, particularly of immunosuppression agent in thegastrointestinal tract. The third step of the presently disclosedprocess for manufacture of presently disclosed formulation involvestreatment of oil entrapped solubilised form of drug with selected amountof one or more of the surfactants. The fourth step of the presentlydisclosed process for manufacture of presently disclosed formulationinvolves treatment of solubilised drug entrapped with oil and one ormore of surfactants with selected amount of the preservative andantioxidant.

In accordance to one of the preferred embodiments of the presentinvention the formulation is made available in a shell, preferably softshell, wherein the said shell essentially comprises of gelatin,glycerin, water and one or more of preservatives, like methyl paraben,propyl paraben, and one or more of colorants, like iron oxide black,titanium dioxide.

In accordance to the preferred embodiment of the present invention, theformulation having presently disclosed composition and prepared inaccordance to the preferred method, as described herein above is filledin the shell, preferably in the soft shell, more preferably in thedisintegrating soft gelatin shell, which becomes part of the formulationfor oral administration and it can not be separate entity of themedicament taken by the patient.

However, the present invention is not restricted by making availabilityof the formulation filled in the presently disclosed shell. It isobvious to those who have knowledge of the art that the presentlydisclosed formulation can also be administered in the form of solutionor after filling in any of the known shell, which may be soft or hardshell. It is further obvious to those who have knowledge of the art thatthe presently disclosed shell can also be used for filling any otherformulation including similar formulation.

Therefore, in accordance to one of the preferred embodiments the presentinvention a disclosure is made of shell essentially comprising ofgelatin, glycerin, water and one or more of preservatives, like methylparaben, propyl paraben, and one or more of colorants, like iron oxideblack, titanium dioxide, wherein gelatin is taken in an amount of isabout 35 to 50% by weight, glycerin is taken in an amount of about 15 to30% by weight, the preservatives, like methyl paraben propyl paraben aretaken in an amount of about 0.2, more preferably of about 0.8% byweight, water is taken in an amount of about 30 to 45% by weight,colorants, like iron oxide black, titanium dioxide are taken in anamount of about 0.5%.

It is obvious from the foregoing description that the advantages of thepresently disclosed selfemulsifiable formulation for oraladministration, as disclosed and described herein above includesincreased solubility, transport rate, bioavailability and bioabsorptionof the immunosuppression agent; capability of making earlybioavailability of the immunosuppression agent, particularly in theupper part of the gastrointestinal tract; capability to meetsatisfactorily the clinical requirements; capability to formmicroemulsions of particle size less than 200 nm, preferably less than100 nm whether administered orally in the form of microemulsions mixedwith fruit juice, milk or any aqueous medium, or administered orallyafter filling in a soft or hard shell; capability to facilitate theavailability of the immunosuppression agent, preferably in the upperpart of the gastrointestinal tract in less than 12 minutes, morepreferably in less than 10 minutes by rupturing the shell; reduced interand intra patient bioabsorption variability; capability to be stored inthe tropical countries for a longer time; capability to formthermodynamically stable oil in water microemulsions in-situ, which arestable for more than 24 hrs and also having capability to beadministered orally after filling in a soft or hard shell, or in theform of solution.

In accordance to the preferred embodiments of the present invention anovel selfemulsifiable formulation having enhanced bioabsorption,bioavailability and immunosuppression activities and comprising of anovel lipophilic system consisting of medium chain triglyceride ofcaprylic acid and capric acid (labrafac lipophile) and labrasol, whereinlabrasol also serves as surfactant, which is combined with otherselected surfactants, like cremophore RH 40 and/or polysorbate 80 isdisclosed, wherein said formulation also comprises of animmunosuppression agent, hydrophilic agent antioxidant and preservative,wherein said immunosuppression agent is essentially cyclosporine havingimmunosuppression activity and said hydrophilic agent is preferablyethanol, said antioxidant is preferably alpha-tocopherol and saidpreservative is preferably benzyl alcohol, and said formulation isprepared by dissolving immunosuppression agent in hydrophilic agentfollowed by entrapping of solubilised immunosuppression agent withlipophilic agent and subsequent treatment with one or more ofsurfactants, and preservative and antioxidant, and is filled insoft-gelatine shell, which is capable of rupturing preferably in lessthan 10 minutes to deliver the formulation in upper part ofgastrointestinal tract, wherein it forms thermodynamically stable oil inwater microemulsions in-situ to have enhanced bioavailability andbioabsorption of the immunosuppression agent, which can show itsenhanced immunosuppression activities thereby.

EXPERIMENTS

The presently disclosed formulations were prepared in accordance to thepreferred method of preparation, as described herein above, and phasediagrams (FIG. 1), relative concentrations of various phases, i.e. oilphase, water phase and mixture of surfactants (FIG. 2), ruptureidentification test in dissolution medium by analysing the percent drugcontent at different time intervals (FIG. 3), percent amount ofnormalised particle size (FIG. 4) and mean plasma concentration of thedrug (cyclosporine) after administration of the shell (FIG. 5) werestudied/carried out in case of selected preparations XIII, XIV and/orXV.

It was observed that all formulations were observed to be clearsolutions, some formulations were slightly turbid in contact withaqueous media but when subjected to particle size analysis, passes themicroemulsion properties. The formulations were filled in soft gelatinshell of the present invention.

The formulations were prepared having compositions as given in FIG. 6.For experimental purpose only different combinations of said agents wereused. However, the combination of experiments XIII, XIV and XV,particularly of XIV and XV were observed to better and were according tothe preferred embodiments of the present invention. These experimentalformulations were subjected to microemulsion tests. From theseexperiments it was observed that presence of labrafac lipophile aslipophile agent in combination with other selected-agents improves themicroemulsion quality significantly.

Now referring to accompanying figures, the FIG. 1 shows the two way plotfor cyclosporine microemulsion—phase behaviour. These phases are mixtureof surfactants (I), oil phase (II) and water phase (III). Point 1represents water in oil microemulsion existence, while point 2represents oil in water microemulsion existence part and point 3 iscoarse emulsion part while point 4 is micelle phase. According to thisphase diagram, the oil phase (II) contains 10% of cyclosporine-Adissolved in hydrophilic agent and then entrapped in oil. The oil phase(II) concentration increases from 0% along the left-hand margin to 100%as shown by arrow. The concentration of aqueous phase (III) increasefrom 0% along the right hand margin to 100% as shown by arrow, while theconcentration of surfactants mixture (I) increase from 0% at the baseline of the plot to 100% as shown by arrow. The relative portion of oil,surfactants and water phases will suitably lie with the area (2), i.e.microemulsion existence field as shown in the FIG. 1. All theexperiments were carried out in the laboratory at a temperature lessthan 25° C. having relative humidity less than 60%.

The FIG. 2 shows three way plot for cyclosporine microemulsion. Thisfigure represents the relative concentration of oil phase (II), waterphase (III) and mixture of surfactants (I). The point A represents thepreferred concentration of microemulsion that is oil phase (II), waterphase (III) and surfactant phase (I).

The FIG. 3 represents the comparative dissolution profile of experimentsXIII, XIV, XV and standard experiment for which Sandimmun Neoral wastaken as standard. The X-axis represents time in minutes required torupture the shell as USP-specification for cyclosporine shells. Therupture time was identified by content analysis in the dissolutionfluid. The Y-axis represents the percentage of cyclosporine dissolved indissolution fluid, which was analysed by high performance liquidchromatography (HPLC).

In view of stringent dissolution test specification of cyclosporinecapsule USP in USP/NF 24 that each capsule should rupture within 15minutes, the presently disclosed formulation of immunosuppression agent,particularly cyclosporine was also subjected to dissolution tests as perUSP specifications and tested for rupture test and shell stability test.The soft gelatin shells having following compositions were used in fourset of experiments for these studies:—

Experiment No. A B C D Ingredient Composition Gelatin 35% 40% 45% 45%Glycerine 15% 25% 25% 20% Sorbitol 18.7% — — — Propyl Paraben  0.04% 0.04%  0.04%  0.04% Methyl Paraben  0.76%  0.76%  0.76%  0.76%Colorants  0.5%  0.5%  0.5%  0.5% Water 30.0% 33.7% 28.7% 33.7%

The soft gelatin shells having above compositions were prepared inaccordance to the preferred method of the present invention and werecured at lower humidity and temperature for less than 25° C. forsufficient time. The cured shells were subjected to rupture test. Thein-vitro dissolution conditions were maintained as follows:—

Apparatus USP Type-II (paddle) Medium Water (500 ml) Rotation per minute(RPM) 50 Temperature of medium 37° C. Time 15 mins

As per USP dissolution test of cyclosporine, it mentions the rupture ofthe shell should be within 15 mins. In the present invention, theefforts were taken not only to comply with the dissolution but also toquantify the rupture time, which gives clear identification of therupture. The rupture identification tests have been designed and wereperformed in dissolution medium by analysing the percent drug content atdifferent time intervals, viz 8, 12 and 15 minutes by HPLC). The percentdrug contents for each rupture time interval are given in FIG. 3, asdescribed herein above. The percent drug contents in case of experimentsXIII, XIV, XV and standard (Sandimmun Neoral) were observed to be 30,31.8, 35 and 0 respectively at time interval of 8 mins, and 75, 70.86,75 and 28.73 respectively at time interval of 12 mins, and 90, 86.83, 95and 91.01 respectively at time interval of 15 mins. This data clearlyshows that the shell of the present invention releases higher amount ofthe drug as compared to the standard.

The FIG. 4 represents percent amount of normalised particle size inexperiment XV, the best mode of the present invention. The X-axisrepresents the particle diameter in nm and Y-axis represents the amountof normalised particle size in percent. It is observed from the figurethat the mean particle size is less than 100 nm.

The FIG. 5 represents the mean plasma concentration of cyclosporineafter administration of shells of the present invention of experimentXV. The X-axis represents the time in hours and Y-axis represents meanplasma concentration in ng/ml. It is observed from the figure that themean plasma concentration that is the bioavailability of the drug ismore than the standard at the early part of the gastrointestinal tractand having the less variability in the inter and intra patient for thedose response.

The presently disclosed formulation (experiment XV) was subjected tobioequivalence study and the results were compared with thestandard—Sandimmun Neoral. The bioequivalence study of formulation ofexperiment XV, best mode of performing the present invention, wascarried out on 24 healthy male subjects using 50 mg of sample of thispreparation and the results were compared with 50 mg of sample ofSandimmun Neoral. All the subjects were adult, healthy, non-smokingmales. The mean (±S.E.) age and weight for the subjects were 27.42±0.97years and 63.63±1.38 kg respectively. Subjects were selected for theparticipation in the study after providing informed written consent andsuccessfully completing a battery of medically related examinationsincluding medical history, complete physical examination,electrocardiogram and a laboratory profile with hematological, urine andbiochemical tests. Subjects were excluded if they had received any drugthat are known to induce the drug-metabolising enzymes within threemonths of study entry. History of hypersensitivity to any drug was ruledout. In a randomized cross over and comparative study designed, 24subjects received these two preparations on two occasions with a washout period of two weeks. Subjects reported to the test facility in theevening before drug administration. No food was permitted for at least10 hrs before the administration of the drug. Next morning afterattending to the morning routine, subjects were made to lie to supine.An indwelling teflon needle was introduced in the left fore arm vein andfasting blood sample was collected. Two shells of either formulationswere administered with 240 ml of water. Blood was collected incentrifuge tube containing 0.1 ml of 10% ETDA. Post dose sampling timeafter drug administration were 0.50, 1.00, 1.50, 2.00, 2.50, 3.00, 3.50,4.00, 5.00, 6.00, 8.00 10.00, 12.00, 24.00 and 48.00 hrs. Blood sampleswere centrifuged in cooling centrifuge, maintained at −20° C.±5° C.;with appropriate labels, identifying subject numbers, study day and timeof blood collection. Fluid intake was controlled and consistent for thefirst four hours following drug administration as follows: drug wasadministered with 240 ml of water, 280 ml of a non-caffeine containingsoft drink provided 4.0 hrs post dose. Water was allowed ad libitumthere after. Standarised breakfast, lunch and dinner were served to thesubjects at 4.0, between 7 to 8 hrs and 14 hrs respectively. Emergenceof symptoms, if any were noted by the subjects at the end of the studyin the symptom check list formed. The subjects were housed in thelaboratory for the entire period. It was randomised comparative studywith a two way cross over design. Plasma cyclosporine levels weremeasured by HPLC method. The drug was extracted from plasma and injectedon HPLC system. The chromatography was carried out on C₁₈ column usingacetonitrile:distilled water in the ratio of 70:30 (v/v) at flow rate of1.0 ml per min at 80° C. The detection was carried out using aUV-detector. The lowest limit of qualification of the drug from plasma20 ng/ml.

Administration of standard formulation (Sandimmun Neoral) showed amiximum concentration of cyclosporine 218.2±16.9 ng/ml in plasma(C_(max)) (ln C_(max) 5.2979±0.0936) at 2.42±0.16 hrs (T_(max)), whilethat of test formulation (Experiment XV) showed a maximum concentrationof cyclosporine 208.5±12.8 ng/ml (C_(max)) (ln C_(max) 5.2912±0.0673) at2.29±0.11 hrs (T_(max)).

The AUC_((0-t)) for standard formulation was 518.35±50.15 ng/ml×hr (lnAUC_((0-t)) 6.1406±0.1010) and for test formulation, it was 518.71±46.97ng/ml×hr (ln AUC_((0-t)) 6.1238±0.1163).

The AUC_((0-∞)) for standard formulation was 611.64±54.55 ng/ml×hr (lnAUC_((0-t)) 6.3262±0.0897) and for test formulation, it was 597.27±50.13ng/ml×hr (in AUC_((0-∞)) 6.2847±0.1062).

The elimination rate constants for standard and test formulations were0,140±0.023 hr⁻¹ and 0.168±0.028 hr⁻¹ and elimination half-lives were7.98±0.97 hrs and 7.34±1.44 hrs respectively.

In this study, with both the formulations, standard and experiment XV,cyclosporine was detected in plasma in few subjects at 0.50 hrs afteringestion of formulation. Cyclosporine was detected up to 8 hrs in someof the subjects post-dose with both the formulations. C_(max) values,time at which they were achieved T_(max) were comparable with both theformulations, so also were AUC_((0-t)), AUC_((0-∞)), K_(el) and T_(1/2).When ANOVA was applied with the subjects, period and treatment asvariables no significant variation was observed for T_(max), whereassubject parameter was found significant for C_(max), ln C_(max),AUC_((0-t)), ln AUC_((0-t)), AUC_((0-∞)) and ln AUC_((0-∞)).

The 90% confidence interval for cyclosporine for the C_(max),AUC_((0-t)) and AUC_((0-∞)) values were 87.26% to 104.34%, 84.10% to117.23% and 81.76% to 114.60% respectively. For the log-transformed datathey were 90.54% to 109.69%, 82.24% to 118.00% and 80.21% to 115.57%respectively. The ratio of the least squares means of the C_(max)AUC_((0-t)) and AUC_((0-∞)) of the test/standard were 95.56%, 100.07%and 97.65% respectively. For the log-transformed data the ratios were99.87%, 99.73% and 99.34% respectively.

The power of test for cyclosporine for the C_(max), AUC_((0-t)) andAUC_((0-∞)) value was 97.44% and for log-transformed data it was 91.15%.The inter-subject variability for cyclosporine for C_(max), AUC_((0-t))and AUC_((0-∞)) values were 17.54%, 33.18% and 33.31% respectively. Forlog-transformed data the values were 19.46%, 37.51% and 37.98%respectively. When AUC_((0-t)) of both the formulations were compared,experiment XV and Standimmun Neoral showed bioavailability of 100.07%.

The bioequivalence data of both the formulations—standard and experimentXV using 50 mg shells is given below. The mean (ng/ml), S.D., S.E. andCOV (%) were measured at time intervals of 0.00, 0.50, 1.00, 1.50, 2.00,2.50, 3.00, 3.50, 4.00, 5.00, 6.00, 8.00 10.00, 12.00, 24.00 and 48.00hrs.

Sandimmun Neoral Experiment XV Time in Mean COV Mean COV hrs (ng/ml)S.D. S.E. (%) (ng/ml) S.D. S.E. (%) 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 0.50 19.1 56.4 11.5 295.81 22.8 42.0 8.6 184.41 1.00 66.7 95.319.5 142.81 71.8 51.4 10.5 71.58 1.50 137.2 92.1 18.8 67.13 121.0 62.112.7 51.34 2.00 150.6 77.5 15.8 51.50 160.6 74.2 15.1 46.17 2.50 163.478.4 16.0 48.01 164.9 62.5 12.8 37.89 3.00 159.8 92.2 18.8 57.72 157.272.6 14.8 46.17 3.50 114.7 57.6 11.8 50.25 109.8 64.7 13.2 58.96 4.0075.3 62.7 12.8 83.27 73.2 58.1 11.9 79.41 5.00 37.9 39.6 8.1 104.54 42.343.9 9.0 103.96 6.00 21.4 27.9 5.7 130.08 19.0 25.6 5.2 134.78 8.00 4.610.7 2.2 232.42 7.0 14.3 2.9 203.34 10.00 1.2 5.7 1.2 489.71 0.0 0.0 0.00.0 12.00 1.1 5.5 1.1 490.04 0.0 0.0 0.0 0.0 24 0.0 0.0 0.0 0.0 0.0 0.00.0 0.0 48 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 AUC_((0−t)) 518.35 245.7550.16 47.41 518.72 230.14 46.98 44.37 AUC_((0−t)) 611.64 267.32 54.5743.70 597.27 245.61 50.14 41.12

The mean and ±SEM of both the preparations for C_(max) (ng/ml), lnC_(max), T_(max), AUC_((0-t)) (ng/ml*hr), ln AUC_((0-t)), AUC_((0-∞))(ng/ml*hr), ln AUC_((0-∞)), T_(1/2) hr and K_(el)/hr are given below:

Preparation Sandimmun Neoral Experiment XV Parameter Mean ± SEM Mean ±SEM C_(max) 218.2 16.9 208.5 12.8 ln C_(max) 5.2979 0.0936 5.2912 0.0673T_(max) 2.42 0.16 2.29 0.11 AUC_((0−t)) 518.35 50.15 518.71 46.97 lnAUC_((0−t)) 6.1406 0.1010 6.1238 0.1163 AUC_((0−∞)) 611.64 54.55 597.2750.13 ln AUC_((0−∞)) 6.3262 0.0897 6.2847 0.1062 T_(½) 7.98 0.97 7.341.44 K_(el) 0.140 0.023 0.168 0.028

The above bioequivalence data shows that the trial of composition offormulation of experiment XV has less percentage of Coefficient ofVariation (COV) and standard error (SE) than the standard formulation.From this data, it can be concluded that the presently disclosedformulation shows significantly less variability in inter and intrapatient dose response than the standard formulation, which is a uniquecharacteristic required for the formulation, particularly for thecyclosporine formulation microemulsions. From the above bioequivalencedata, one another advantage of the presently disclosed formulation whichis apparent, is that the soft gelatin shell having very low rupturetime, that is, about 8 mins, will give fast release of the drug out ofthe formulation for the immediate bioavailability of the drug, that is,of the immunosuppression agent, that is, of the cyclosporine, i.e. 22.8ng/ml in 30 mins as against standard formulation i.e. 19.1 ng/ml.

1. A selfemulsifiable formulation for oral administration, wherein saidformulation is consisting essentially of immunosuppression agent,hydrophilic agent, lipophilic agent, surfactants, antioxidant andpreservative, wherein said immunosuppression agent is cyclosporine-Ahaving immunosuppression activity and is taken in an amount of about 2to 10% by weight of the formulation; said hydrophilic agent isconsisting essentially of ethanol taken in a weight ratio of about 1:0.5to 1:2.5 with respect to said immunosuppression agent; said lipophilicagent is medium chain triglycerides of caprylic and capric acids takenin a ratio of immunosuppression agent to lipophilic agent of about 1:1.5or about 1:1.7 by weight; said surfactants are combination ofpolyglycolised transesterified caprylic and capric glycerides having ahydrophilic lipophilic balance (HLB) of about 14 and a saponificationvalue of about 85 to 105, polysorbate 80 and cremophor RH 40; saidantioxidant is selected from the group consisting of alpha-tocopherolascorbyl palmitate, butyl hydroxy anisole, butyl hydroxy toluene, andpropyl gallate; and said preservative is selected from one of ethanoland benzyl alcohol wherein said formulation forms microemulsions ofparticle size less than 200 nm on contact with gastric juice or aqueousmedium.
 2. A selfemulsifiable formulation, as claimed in claim 1,wherein said medium chain triglyceride of caprylic acid and capric acidhas specific gravity of about 0.93 to 0.96, refractive index of about1.44 to 1.46, acid value less than about 0.2, saponification value ofabout 310 to 360, iodine value less than about 1 and water content lessthan about 0.5.
 3. A selfemulsifiable formulation, as claimed in claim1, wherein said immunosuppression agent is taken in an amount of about 5to 10%.
 4. A selfemulsifiable formulation, as claimed in claim 1,wherein said immunosuppression agent and hydrophilic agent are taken inratio of about 1:0.5 to 1:2.5, or of about 1:1.25 by weight.
 5. Aselfemulsifiable formulation, as claimed in claim 1, wherein saidpolyglycolised transesterified caprylic and capric glycerides(Labrasol®) is taken in the ratio of about 2:1, or of about 3:1, or ofabout 6:1 by weight with respect to lipophilic agent.
 6. Aselfemulsifiable formulation, as claimed in claim 1, wherein saidpolyglycolised transesterified caprylic and capric glycerides(Labrasol®) and cremophor RH 40 are taken in the ratio of about 1:1, orof about 2.5:1 by weight, and said “polyglycolised transesterifiedcaprylic and capric glycerides (Labrasol®) and polysorbate 80 are takenin the ratio of about 2:1, or of about 4.5:1 by weight.”
 7. Aselfemulsifiable formulation, as claimed in claim 1, wherein saidpolyglycolised transesterified caprylic and capric glycerides(Labrasol®), polysorbate 80 and cremophor RH 40 are taken in the ratioof about 4:1:1.5 by weight respectively.
 8. A selfemulsifiableformulation, as claimed in claim 1, wherein said preservative is benzylalcohol and is taken in the amount of about 0.5 to 1% by weight.
 9. Aselfemulsifiable formulation, as claimed in claim 1, wherein saidantioxidant is alpha-tocopherol and is taken in the amount of about0.00007 to 0.00009%.
 10. A selfemulsifiable formulation, as claimed inclaim 1, wherein said formulation forms microemulsions of particle sizeless than 100 nm on contact with gastric juice or aqueous medium.
 11. Amethod of preparation of selfemulsifiable formulation, said formulationconsisting essentially of immunosuppression agent, hydrophilic agent,lipophilic agent, surfactants, antioxidant and preservative, whereinsaid immunosuppression agent is cyclosporine-A having immunosuppressionactivity and is taken in an amount of about 2 to 10% by weight of theformulation; said hydrophilic agent is consisting essentially of ethanoltaken in a weight ratio of about 1:0.5 to 1:2.5 with respect to saidimmunosuppression agent; said lipophilic agent is medium chaintriglycerides of caprylic and capric acids taken in a ratio ofimmunosuppression agent to lipophilic agent of about 1:1.5 or about1:1.7 by weight; polysorbate 80, and cremophor RH 40″ has been amendedto “said surfactants are combination of polyglycolised transesterifiedcaprylic and capric glycerides having a hydrophilic lipophilic balance(HLB) of about 14 and a saponification value of about 85 to 105,polysorbate 80 and cremophor RH 40” said antioxidant is selected fromthe group consisting of alpha-tocopherol ascorbyl palmitate, butylhydroxy anisole, butyl hydroxy toluene, and propyl gallate; and saidpreservative is selected from one of ethanol and benzyl alcohol whereinsaid formulation forms microemulsions of particle size less than 200 nmon contact with gastric juice or aqueous medium and wherein said methodcomprising the following steps;— a) dissolution of immunosuppressionagent in hydrophilic agent, b) entrapping of solubilisedimmunosuppression agent with lipophilic agent, c) treatment of oilentrapped solubilised form of drug with combination of surfactants, d)treatment of solubilised drug entrapped with oil and combination ofsurfactants with preservative and antioxidant.
 12. A selfemulsifiableformulation, as claimed in claim 1, wherein said formulation is madeavailable in a soft shell, wherein said shell is consisting essentiallyof gelatin, glycerin, water and one or more of preservatives, selectedfrom the group consisting of methyl paraben, and/or propyl paraben, andone or more of colorants, such as iron oxide black or titanium dioxide.13. A selfemulsifiable formulation, as claimed in claim 12, wherein saidgelatin is taken in an amount of about 35 to 50% by weight.
 14. Aselfemulsifiable formulation, as claimed in claim 12, wherein saidglycerin is taken in an amount of about 15 to 30% by weight of theshell.
 15. A selfemulsifiable formulation, as claimed in claim 12,wherein the preservatives are taken in an amount of about 0.2%, or ofabout 0.8% by weight of the shell.
 16. A selfemulsifiable formulation,as claimed in claim 12, wherein water is taken in an amount of about 30to 45% by weight of the shell.
 17. A selfemulsifiable formulation, asclaimed in claim 12, wherein said colorant is taken in an amount ofabout 0.5% by weight of the shell.
 18. A selfemulsifiable formulation,as claimed in claim 1, wherein said formulation is administered forimmunosuppression therapy.